CA2360488A1 - Use of a plant extract of the genus rosmarinus in compositions for treating ageing skin symptoms - Google Patents

Abstract

The invention concerns the use of at least a plant extract of at least the genus Rosmarinus, in or for the preparation of a composition for preventive and/or curative treatment of the ageing skin symptoms. In particular the extract or the composition are designed to stimulate the restoring of the skin structure and/or to inhibit degeneration of the skin and/or the mucous membranes by stimulating collagen synthesis and/or by inhibiting collagenase.

Description

WO 00/41674, PCT / FR00 / 00034 Use of an extract from a plant of the genus Rosmarinus in compositions for treating skin signs of aging The invention relates to the use of at least one extract of at least one vegetal of the genus Rosmarinus in or for the preparation of a composition intended for to treat, in a preventive and / or curative way, the cutaneous signs of aging.
In particular, the extract or the composition are intended to stimulate the restructuring and / or inhibit the degradation of the skin and / or mucous membranes by stimulation of collagen synthesis and / or by inhibiting collagenases. The invention at also relates to a cosmetic treatment process for the skin and / or mucous membranes.
Human skin consists of two compartments, namely a compartment superficial, the epidermis, and a deep compartment, the dermis.
The natural human epidermis is mainly composed of three types of cells which are the majority keratinocytes, the melanocytes and the cells of Langerhans. Each of these cell types contributes by its functions clean to the essential role played in the body by the skin.
The dermis provides the epidermis with solid support. It is also its element nurturer. It mainly consists of fibroblasts and a matrix extracellular itself composed mainly of collagen, elastin and of a substance, called the fundamental substance, components synthesized by the fibroblast. There are also leukocytes, mast cells or tissue macrophages. It is also crossed by blood vessels and nerve fibers. In normal skin, i.e. non-pathological or scar, the fibroblast is in a quiescent state, i.e. no proliferative, little active from a metabolic point of view and not mobile.
It is the collagen fibers which ensure the solidity of the dermis. Fibers of collagen are made up of fibrils sealed to each other, forming so more than ten different types of structures. The solidity of the dermis is in big part due to the entanglement of collagen fibers packed against each other the others in every way. Collagen fibers participate in elasticity and the tone of the skin and / or mucous membranes.
The collagen fibers are constantly renewed but this renewal decreases with age which leads to thinning of the dermis. This

2 - thinning of the dermis is also due to pathological causes such as for example hypersecretion of corticosteroid hormones, certain diseases (Marfan syndrome, Ehlers-Danlos syndrome) or deficiencies vitamin (scurvy). It is also recognized that extrinsic factors such as ultraviolet rays, tobacco or certain treatments (Glucocorticoids, vitamin D and derivatives for example) also have an effect on the skin and his collagen levels.
However, various factors cause the breakdown of collagen, with all the l0 consequences which one can envisage on the structure and / or firmness of the skin and / or mucous membranes.
Although very resistant, collagen fibers are sensitive to certain enzymes called collagenases. Degradation of collagen fibers causes the appearance of soft, wrinkled skin than humans, preferring the appearance of smooth, taut skin has always sought to combat.
Collagenases are part of a family of enzymes called metalloproteinases (MMPs) which are themselves members of a family of enzymes 2o proteolytics (endoproteases) which have a zinc atom coordinated to 3 cysteine and methionine residues in their active site and which degrade macromolecular components of the extracellular matrix and laminae basal at neutral pH (collagen, elastin, etc.). Very widespread in the living world, these enzymes are present, but weakly expressed, in normal physiological situations like organ growth and tissue renewal.
Their overexpression in humans and their activation are however linked to many processes, sometimes pathological, which involve the destruction and matrix remodeling. This results in either an uncontrolled absorption of the 3o extraceflular matrix, or conversely the installation of a state of fibrosis.
The metalloproteinase family consists of several well-defined groups defined based on their similarities in terms of structure and specificity of substrate (see Woessner JF, Faseb Journal, vol. 5, 1991, 2145). Among these groups, we may cite collagenases intended to degrade fibrillar collagens (MMP-1 or interstitial collagenase, MMP-8 or neutrophil collagenase, MMP-13 or collagenase 3), gelatinases which degrade type IV collagen or any form of denatured collagen (MMP-2 or gelatinase A (72 kDa), MMP-9 or gelatinase B (92 kDa)), stromelysins (MMP-3) whose broad spectrum of activity

3 targets proteins in the extracellular matrix such as glycoproteins (fibronectin, laminin), proteoglycans, etc., or membrane metalloproteinases.

WO 00/4167

4 PCT / FR00 / 00034 Prolonged exposure to ultraviolet radiation, particularly ultraviolet radiation type A and / or B, has the effect of stimulating expression of collagenases, particularly MMP-1. This is one of the components of photoinduced skin aging.
In addition to the menopause, the main modifications concerning the dermis are a decrease in collagen level and dermal thickness. That causes menopausal women to thin their skin and / or mucous membranes. The woman then feels a feeling of "dry skin" or skin which pulls and there is an accentuation of fine lines and wrinkles area. The skin has a rough appearance on palpation. Finally the skin presents a decreased flexibility.
We then understand from reading the above the importance of collagen in tissue structure, particularly of the skin and / or mucous membranes, and the importance of not only maintaining the rate in the tissues but also to fight its degradation so to fight against aging it either chronobiological or photo-induced and its consequences, the thinning of the dermis and / or breakdown of collagen fibers which cause appearance of soft, wrinkled skin.

One of the aims of the present invention is therefore to be able to have a product new which has a stimulating effect on collagen synthesis, a effect collagenase inhibitor and if possible no significant side effects.
Now, surprisingly and unexpectedly, the plaintiff has now discovered that an extract from at least one plant of the genus Rosmarinus has a stimulating activity of collagen synthesis and inhibiting activity of.
collagenase activity.
Plants of the genus Rosmarinus, in addition to their taste and fragrant, are known for their anti-inflammatory effects. We can cite to this with regard to patent applications FR-A-2504551, JP = A-7017846, WO-A-9325209, SU-A-1733000, FR-A-2662078, DE-A-3536342, US 5393526 or the application filed by the applicant under number WO-A-9701288.

To the knowledge of the applicant, the stimulating activity of the synthesis of collagen and the inhibitory activity of the collagenase activity of a extract from at least one plant of the genus Rosmarinus has never been described.

5 Thus, the primary object of the invention is the use of at least one extract at least a plant of the genus Rosmarinus in or for the preparation of a composition, the extract or composition being intended to treat, preventively and or healing, skin signs of aging.
lo By extract of at least one plant of the genus Rosmarinus, is understood as well a raw mixture of coarsely chopped parts of the vegetable and extraction solvent as elaborate preparations of active ingredients solubilized during extraction.
The term active ingredient means any molecule or composition capable of modify or modulate the functioning of at least one biological system given.
By cutaneous signs of aging is understood all modifications of appearance 2o outside of the skin due to aging, whether chronobiological and / or photo-induced, such as fine lines and wrinkles, withered skin, mole skin, the thinned skin, lack of elasticity and / or skin tone, but also all internal skin changes that do not translate systematically by a modified external appearance, such as for example all internal damage to the skin, particularly collagen, consecutive at exposure to ultraviolet radiation.
A second object of the invention is the use of at least one extract of at least a vegetable of the genus Rosmarinus in or for the preparation of a composition, the extract or the composition being intended to stimulate synthesis of the collagen.
The third object of the invention is the use of at least one extract of minus one vegetable of the genus Rosmarinus in or for the preparation of a composition, the extract or the composition being intended to inhibit the expression of proteases of the extracellular matrix, particularly metalloproteinases and again more particularly metalloproteinase type 1.

6 A fourth object of the invention is the use of at least one extract of minus one vegetable of the genus Rosmarinus in or for the preparation of a composition, the extract or the composition being intended to treat cutaneous lesions related to menopause.
A fifth object of the invention is the use of at least one extract of minus one vegetable of the genus Rosmarinus in or for the preparation of a composition, the extract or the composition being intended to combat wrinkles and fine lines.
l0 The sixth object of the invention is the use of at least one extract of minus one vegetable of the genus Rosmarinus in or for the preparation of a composition, the extract or the composition being intended to fight against withered skin.
A seventh subject of the invention is the use of at least one extract of minus one vegetable of the genus Rosmarinus in or for the preparation of a composition, the extract or the composition being intended to fight against soft skin.
The invention has as its eighth object the use of at least one extract of minus one vegetable of the genus Rosmarinus in or for the preparation of a composition, the extract or the composition being intended to fight against thinned skin.
The ninth object of the invention is the use of at least one extract of minus one vegetable of the genus Rosmarinus in or for the preparation of a composition, the extract or composition being intended to combat the lack elasticity and / or of skin tone.
The extract of at least one plant of the genus Rosmarinus used according to the invention can be obtained from plant material from whole plant or part of plant like leaves, stems, flowers, petals, roots or again 3o dedifferentiated cells.
By dedifferentiated plant cells is meant any plant cell that does not having none of the characteristics of a particular specialization and capable of live on its own and not in dependence on other cells.
Preferably according to the invention, the whole plant is used, particularly stem and / or leaves, very particularly leaves.

7 The extract of at least one plant of the genus Rosmarinus can be any extract prepare from any plant material from at least one plant of the genus Rosmarinus grown in vivo or derived from in vitro culture.
By in vivo culture is meant any culture of conventional type, that is to say in ground to in the open air or in a greenhouse, or even above ground.
By in vitro culture is meant all of the techniques known to man of profession which allows artificially obtaining a plant or a part lo of a plant. The selection pressure imposed by the physical-chemicals during the growth of plant cells in vitro allows to get a standardized plant material available throughout the year in contrary plants grown in vivo.
Preferably according to the invention, a plant of the genus Rosmarinus is used.
from in vivo culture.
The Rosmarinus officinale species has two varieties which are Rosmarinus tenuifolius and Rosmarinus latifolius.
The extract of the invention can be prepared from plant material derived from the species Rosmarinus officinales whatever the variety.
Any extraction method known to those skilled in the art can be used to prepare the extract according to the invention.
Mention may, in particular, be made of alcoholic extracts, in particular ethanolic or still hydroalcoholic.
Preferably according to the invention, the extract is an aqueous extract.
It is also possible to use an extract prepared by the method described in the French patent application No. 95-02379 filed by the applicant.
So, in a first step we grind the plant material in a solution aqueous in cold and in a second stage the particles in suspension are removed from the aqueous solution from the first step. This solution aqueous corresponds to the extract.
Optionally, in a third step, the aqueous solution is sterilized from the second step.
This extract can then be lyophilized.

8 The first step can advantageously be replaced by an operation of simple freezing of plant tissues (for example at -20 ° C or even at -180 ° C
in liquid nitrogen), followed by an aqueous extraction using the second and third steps described above.
i0 The cold treatment makes it possible to freeze the enzymatic activities, the filtration sterilizing prevents the degradation of active ingredients by microorganisms the environment. Finally, the water vehicle is compatible with ex receivers vivo and facilitates cosmetic or pharmaceutical formulations.
It is known that plant extracts contain responsible oxidases, inter alia, the oxidation of said extracts. Now such an oxidation leads to one dark brown coloring of the extracts and a pungent odor making them little compatible with their use in cosmetics. In this order of idea we know is in particular a laccase whose molecular weight is greater than 100,000 daltons.
Thus, advantageously, the extract obtained can be fractionated by any method of known fractionation making it possible to eliminate the oxidases and in particular the 2o polyphenoloxidase. We can for example filter the extract of the invention on a dialysis membrane to remove molecules of molecular weight greater than 100,000 daltons. It is also possible to subject extract one fractionation by selective precipitation.
25 Other methods make it possible to guard against oxidation phenomena. In in particular, the extract can also be stabilized. Any method of stabilization known can be used according to the invention. We can for example stabilize the extract of the invention by bubbling nitrogen through to remove dissolved oxygen or else in adding cysteine and / or sulfur derivatives to a concentration final 30 between 0.5 g / l and 10 g / l and preferably between 1 g / l and 2.5 g / l.
Obviously the extract according to the invention can be fractionated and stabilized.
The extract alone can constitute the active principle of the compositions of the invention.
In a particular form of the invention, the extract does not contain acid rosmarinic.

9 An example of preparation of extract usable according to the invention is given by elsewhere in the examples.
The amount of extract usable according to the invention is of course a function of the effect sought after and can therefore vary to a large extent.
To give an order of magnitude, one can use an extract as defined previously in an amount representing from 0.001% to 20% of the total weight of the composition and preferably in an amount representing from 0.01% to 10%
of the total weight of the composition.
The present invention further relates to a method of treatment cosmetic of the skin intended to stimulate the synthesis of collagen and / or treat reached related to age and / or menopause and / or fight against thinning of dermis and / or combat the appearance of soft and / or wrinkled skin, characterized speak applied to the skin, the hair, and / or the mucous membranes, a cosmetic composition comprising at least extract of at least one plant from genus Rosmarinus.
The cosmetic treatment process of the invention can be implemented in particular by applying the cosmetic compositions as defined above above, according to the usual technique of use of these compositions. Through example: application of creams, gels, serums, lotions, milks, shampoos or ante-sun compositions, on the skin, or on the hair or further application of toothpaste on the gums.
The following examples and compositions illustrate the invention without limiting it not at all. In the compositions the proportions indicated are percentages by weight.
Example 1: Preparation of an extract of Rosmarinus officinale 636 grams of Rosmarinus officinale plant material, aerial part corresponding to stems and leaves, are immersed in about 1 kg nitrogen liquid.
After removing the stems, the leaves are reduced to powder by grinding at Turrax at 24,000 rpm. for 1 minute at 4 ° C (ice bath).
The powder obtained is mixed with 5 liters of 0.05 M phosphate buffer at pH 8.5.
The whole is stirred for 30 minutes at 4 ° C., then centrifuged at 10,000 G
at 4 ° C.

The supernatant is filtered at 0.22 Nm (sterilizing filtration).
The extract is then fractionated by ultrafiltration on a membrane of the type Sartorius so to eliminate oxidation phenomena.
The extract is then lyophilized. 29.5 grams of extract are thus obtained active 5 called "lyophilized extract".
Example 2 Effect of the extract of Example 1 on the synthesis of collagen The study is made by measuring the incorporation of radioactive proline in of cultures of normal hurricane dermal fibroblasts.

10 The fibroblast cultures are carried out according to conventional methods of cell culture, namely in MEM / M199 medium sold by the company Gibco, in presence of sodium bicarbonate (1.87 mg / ml) of L-glutamine (2 mM), penicillin (50 IU / ml) and 10% fetal calf serum (Gibco).
The test is carried out on cell cultures at 80% confluence in plate of 24 holes. The extract of Example 1 (lyophilized extract titrated at 25 mg / ml of water for obtain a 100% stock solution), at final concentrations of 0.2%, 1% and 2%, is brought into contact with the cells for 48 hours. Marking with proline tritiated (L- [2,3 3H] -proline sold by Amersham, 33 ~ Ci / ml) is carried out while 24 hours.
The level of incorporated tritiated proline is measured at the end of the test by precipitation protein acid on filters and liquid scintillation counting.
The results are evaluated relative to a control constituted by cells not having been treated with the extract of Example 1.
A positive control (vitamin C at 20 pg / ml) known to stimulate the synthesis of collagen is introduced into the test as a reference.
The results of this test, expressed as activation percentages, are presented in the following table % Cpm processing Untreated cells 7498 - -Vitamin C 13091 75 <0.01 0.2% 9537 8> 0.05 Extract from example 1 1 9181 22> 0.05 %

2% 8098 27> 0.05

11 cpm: strokes per minute p: confidence interval calculated using the Dunett method.
These results show that the extract of Example 1 stimulates in a significant incorporation of proline into collagen and therefore it has an effect on the collagen neosynthesis. .
Example 3 Effect of the extract of Example 1 on the expression of collagenases.
The study makes it possible to evaluate the effectiveness of a product in inhibiting the induction of transcription of the collagenase gene by ultraviolet radiation.
The test is carried out on Hela cells (ATCC CCL2) in which the promoter of the collagenase 1 gene (3.8 kB) cloned upstream of the gene for chloramphenicol acetyltransferase (CAT) was introduced.
The cells are exposed to ultraviolet B (UVB) radiation emitted by a Phifips TL 12W / 20 tube, KADACEL filtered to eliminate UVC, at doses of and 10 mj / cm2. The cells are then brought into contact for 2 hours with the extract of Example 1 at concentrations varying from 0.05% to 0.5%.
The CAT assay is carried out using a "CAT-Elisa" kit sold by the society Boehringer, as directed by the supplier.
"Baseline" of CAT expression is measured in cells having received neither ultraviolet radiation nor treatment with the product to be tested.
The test results, presented in the attached table, are expressed by report at the basic rate (100%), as a percentage of activation or inhibition of expression of the CAT gene.
These results therefore reflect the activity of the collagenase 1 promoter after induction by ultraviolet light and whether or not the product to be tested is present.
UV B * Without extract Extract Extract 0.05% 0.1%

5,926,416,289 *: Dose in mJ / cm2 These results show that - the activity of the collagenase promoter is stimulated by UVB rays (column "without extract");

12 - This stimulation is reduced in the presence of the extract of Example 1 (columns "Extract to ...");
These results clearly show the inhibitory effect of the synthesis of collagenase 1, by inhibiting the activity of its promoter, of Rosmarinus extract officinalis from example 1.
Example 4: Examples of formulations illustrating the invention and especially the compositions according to the invention combining at least one extract of Rosmarinus l0 officinalis. These compositions were obtained by simple mixing of different components.
Composition 1: Make-up removing lotion for the face Extract from Example 1 10.00 Antioxidant 0.05 Isopropanol 40.00 Preservative 0.30 Water qs 100.00 Composition 2: Shampoo Extract from Example 1 0.50 Hydroxypropyicellulose (Klucel H ~ sold by the company Hercules) 1.00 Perfume 0.50 Preservative 0.30 Water qs 100.00 Composition 3: Facial care cream (emulsion, oil in water) Extract from Example 1 2.00 Glycrol Starate 2.00 Polysorbate 60 (Tween 60 ~ sold by the company1.00 HERE) Staric acid 1.40 Trithanolamine 0.70 Carbomer 0.40 Liquid fraction of shea butter 12.00 Perhydrosqualne 12.00 Antioxidant 0.05 Perfume 0.50 Preservative 0.30

13 Water qs 100.00 Composition 4: Gel for the skin Extract from Example 1 0.50 All trans retinoic acid 0.05 Hydroxypropylcellulose (Klucel H ~ sold by the company Hercules) 1.00 Antioxidant 0.05 Isopropanol 40.00 1o Preservative 0.30 Water qs 100.00 Composition 5: Gel for facial care Extract from Example 1 5.00 Hydroxypropylcellulose (Klucel H ~ sold by the company Hercules) 1.00 Antioxidant 0.05 Isopropanol 40.00 Preservative 0.30 Water qs 100.00 Composition 6: Gel Extract from Example 1 1.00 Hydroxypropylcellulose (Klucel H ~ sold by the company Hercules) 1.00 Antioxidant 0.05 Lidocaine hydrochloride 2.00 Isopropanol 40.00 Conservative . 0.30 Water qs 100.00 Composition 7: Care cream for solar arrhythmia (oil-in-water emulsion) Extract from Example 1 2.50 Glycrol Starate 2.00 Polysorbate 60 (Tween 60 sold by the company HERE) 1.00 Staric acid 1.40 Glycyrrhtinic acid 2.00 Trithanolamine 0.70 Carbomer 0.40 Liquid fraction of shea butter 12.00

14 Sunflower oil 10.00 Antioxidant 0.05 Perfume 0.50 Preservative 0.30 Water qs 100.00 Composition 8: Anti-wrinkle care cream for the face (oil / water emulsion) Extract from Example 1 5.00 Glycrol Starate 2.00 l0 Polysorbate 60 (Tween 60 ~ sold by 1.00 company HERE) Staric acid 1.40 N-octanoyl-5-salicylic acid 0.50 Trithanolamine 0.70 Carbomer 0.40 Liquid fraction of shea butter 12.00 Perhydrosqualne 12.00 Antioxidant 0.05 Perfume 0.50 Preservative 0.30 Water qs 100.00 Composition 9: Lotion Extract from Example 1 0.50 Glycolic acid 50.00 Hydroxypropyfcellulose (Klucel H ~ sold by the company Hercules) 0.05 Preservative 0.30 NaOH qs pH = 2.8 Ethanol qs 100.00

Claims (23)

1. Use of at least one extract of at least one plant of the genus Rosmarinus for the preparation of a composition intended to treat, in a preventive manner and or healing, the cutaneous signs of aging. 2. Use of at least one extract of at least one plant of the genus Rosmarinus for the preparation of a composition intended to stimulate the synthesis of collagen. 3. Use of at least one extract of at least one plant of the genus Rosmarinus for the preparation of a composition intended to inhibit the expression of proteases of the extracellular matrix. 4. Use according to claim 3, characterized in that the composition are intended to inhibit the expression of metalloproteinases. 5. Use according to any one of claims 3 or 4, characterized speak fact that the composition are intended to inhibit the expression of the metalloproteinase type 1. 6. Use of at least one extract of at least one plant of the genus Rosmarinus for the preparation of a composition intended to treat skin disorders related to menopause. 7. Use of at least one extract of at least one plant of the genus Rosmarinus for the preparation of a composition intended to combat wrinkles and fine lines 8. Use of at least one extract of at least one plant of the genus Rosmarinus for the preparation of a composition intended to combat flabby skin. 9. Use of at least one extract of at least one plant of the genus Rosmarinus for the preparation of a composition, intended to struggle between flabby skin. 10. Use of at least one extract of at least one plant of the genus Rosmarinus for the preparation of a composition intended to combat thinned skin. 11. Use of at least one extract of at least one plant of the genus Rosmarinus for the preparation of a composition intended to combat the lack of elasticity and/or of skin tone. 12. Use of at least one extract of at least one plant of the genus Rosmarinus for the preparation of a composition intended to fight against the degradations internal the skin following exposure to ultraviolet radiation. 13. Use according to claim 12, the composition being intended for fight against collagen degradation. 14. Use according to any one of the preceding claims, characterized by the fact that the plant material comes from a whole plant or part of plant like the leaves, the stems, your flowers, your petals, the roots or even cells dedifferentiated. 15. Use according to claim 14, characterized in that the material plant comes from the leaves. 16. Use according to any one of the preceding claims, characterized by the fact that the plant material is derived from at least one plant of the genus Rosmarinus grown in vivo or from in vitro culture, 17. Use according to claim 16, characterized in that the material plant is derived from at least one plant of the genus Rosmarinus cultivated in vivo. 18. Use according to any one of the preceding claims, characterized in that the extract is an aqueous extract. 19. Use according to any one of the preceding claims, characterized by the fact that the extract does not contain rosmarinic acid 20. Use according to any one of the preceding claims, characterized in that the extract is used in an amount representing from 0.001% to 20% of total weight of the composition. 21. Use according to the preceding claim, characterized in that as the extract is used in an amount representing from 00.01% to 10% of the total weight of the composition. 22. Use according to any one of the preceding claims, characterized by the fact that the extract is used in the preparation of a composition cosmetic. 23. Method of cosmetic treatment of the skin intended to stimulate the synthesis from collagen and/or treat skin damage related to age and/or menopause and/or combat the thinning of the dermis and/or combat the appearance of the skin soft and wrinkled, characterized by the fact that one applies to the skin, to the hair, and/or on the mucous membranes, a cosmetic composition comprising at least an extract of at least least one plant of the genus Rosmarinus.